Allergy and Immunotoxicology in Occupational Health - The Next Step [1st ed.] 9789811547348, 9789811547355

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Current Topics in Environmental Health and Preventive Medicine

Takemi Otsuki Mario Di Gioacchino Claudia Petrarca   Editors

Allergy and Immunotoxicology in Occupational Health - The Next Step

Current Topics in Environmental Health and Preventive Medicine Series Editor Takemi Otsuki Kawasaki Medical School Kurashiki Okayama, Japan

Current Topics in Environmental Health and Preventive Medicine, published in partnership with the Japanese Society of Hygiene, is designed to deliver well written volumes authored by experts from around the globe, covering the prevention and environmental health related to medical, biological, molecular biological, genetic, physical, psychosocial, chemical, and other environmental factors. The series will be a valuable resource to both new and established researchers, as well as students who are seeking comprehensive information on environmental health and health promotion. More information about this series at http://www.springer.com/series/13556

Takemi Otsuki  •  Mario Di Gioacchino Claudia Petrarca Editors

Allergy and Immunotoxicology in Occupational Health - The Next Step

Editors Takemi Otsuki Department of Hygiene Kawasaki Medical School Kurashiki Okayama Japan

Mario Di Gioacchino Department of Medicine and Aging Science (DMSI) University G. d’Annunzio of Chieti-Pescara Chieti Italy

Claudia Petrarca Department of Medicine and Aging Science (DMSI) University G. d’Annunzio of Chieti-Pescara Chieti Italy

ISSN 2364-8333     ISSN 2364-8341 (electronic) Current Topics in Environmental Health and Preventive Medicine ISBN 978-981-15-4734-8    ISBN 978-981-15-4735-5 (eBook) https://doi.org/10.1007/978-981-15-4735-5 © Springer Nature Singapore Pte Ltd. 2020 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Singapore Pte Ltd. The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721, Singapore

Preface

We published the second volume of this EHPM eBook series in 2017: Allergy and Immunotoxicology in Occupational Health. And this is a new eBook that, 3 years later, describes the next step. As I wrote last time, the three editors will be working together in the Allergy and Immunotoxicology Scientific Committee (AISC) of the International Congress of Occupational Health (ICOH). The ICOH plenary meeting is held once every 3 years and will be held in Melbourne (Australia) in March 2021. Before that it was held in Dublin (Ireland) in 2018. In 2015 it was held in Seoul (Korea), and the previous eBook was published as a mid-term AISC activity between 2015 and 2018. And this time, as a mid-term activity from 2018 to 2021, we decided to write this book again. As in the previous case, we will also introduce new discoveries, following the previous case, regarding basic experimental systems related to allergy and immunotoxicity and immune abnormalities in respiratory disorders (pneumoconiosis) such as silica and asbestos. In addition, we talk about pesticide immunotoxicity—an old and new problem. In addition, this time, a chapter was also set up on regulations on occupational medicine in relation to skin diseases and respiratory diseases. It does not specialize in occupational medicine at present, but also mentions immune reconstitution inflammatory syndrome. In addition, taking into account clinical control and basic experiments, we devoted a number of chapters to nanomaterials to raise awareness of nanomaterials, which is a topic of recent times, as an AISC. Readers of this book who are interested in ICOH, or who are already members of ICOH but did not know about AISC, should definitely stop by the AISC session at the 2021 meeting in Melbourne. I want you. During Dublin in 2018, I had many poster presentations with two symposiums and two oral presentation sessions. Participants from Europe also actively asked questions about basic aspects and controls in the field of occupational medicine. In addition, presentations from Africa were presented at the symposium.

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In particular, from the viewpoint of allergy and immunotoxicity, there is a complex background to pay attention to in the basic occupational health activities and in the health care of workers. Therefore, it is necessary to study carefully on the issues of normal occupational health and health and also to understand the basics of allergology, immunology, and toxicology before responding. And it is also important to take precautionary measures, so further consideration of all AICS members will be important. Among these perspectives, the eBook published in 2017 and the book Allergy and Immunotoxicology in Occupational health: The Next Step are intended to improve the health of readers, all researchers related to occupational health, and even workers. We hope to contribute to disease prevention. Kurashiki, Japan Chieti, Italy  Chieti, Italy 

Takemi Otsuki Mario Di Gioacchino Claudia Petrarca

Introduction

This eBook highlights the importance of allergy and immunotoxicity, especially in occupational medicine. Examples of the substance include nanomaterials, pesticides, fibers, and particulate matter (silicic acid and asbestos). An important viewpoint is respiratory and skin diseases and their clinical regulation or prevention of health disorders due to allergies and immunotoxicity. This year, the second tier of the eBook, published in 2017 as Allergy and Immunotoxicology in Occupational Health, added the phrase “The Next Step” to the title. Also, the three editors are the same as the previous one, but all are active members of the Allergy and Immunotoxicology Scientific Committee (AISC) of the International Congress in Occupational Health (ICOH), and the previous book was ICOH 2015 and 2018 The Next Step will be published as an activity of the AISC during this year’s main competition and this year’s The Next Step. Many thanks to readers who are interested in occupational health and allergies and immunotoxicity.

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Contents

1 Mechanisms of Action of Inhaled Particulates on Allergic Lung Inflammation����������������������������������������������������������������������������������    1 Etsushi Kuroda 2 Metal Nanoparticle Health Risk Assessment����������������������������������������   17 Luca Di Giampaolo, Claudia Petrarca, Rocco Mangifesta, Cosima Schiavone, Cinzia Pini, Alice Malandra, Francesca Bramante, Alessio Pollutri, Michele Di Frischia, and Mario Di Gioacchino 3 Immune Toxicity of and Allergic Responses to Nanomaterials�����������   37 Yasuo Yoshioka, Toshiro Hirai, and Yasuo Tsutsumi 4 Inflammation and Environmental (Ultrafine) Nanoparticles��������������   47 Francesca Larese Filon 5 Monitoring Nanomaterials in the Workplace����������������������������������������   57 Adrienne C. Eastlake, Luca Fontana, and Ivo Iavicoli 6 Immunotoxicity of Nanoparticles ����������������������������������������������������������   75 Claudia Petrarca, Rocco Mangifesta, and Luca Di Giampaolo 7 Occupational Respiratory Allergic Diseases: Occupational Asthma������������������������������������������������������������������������������   95 Sasho Stoleski 8 Occupational Respiratory Allergic Diseases: Occupational Rhinitis������������������������������������������������������������������������������  115 Sasho Stoleski 9 Occupational Skin Diseases��������������������������������������������������������������������  129 Dragan Mijakoski

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10 Expanding Concept of Immune Reconstitution Inflammatory Syndrome: A New View Regarding How the Immune System Fights Exogenous Pathogens������������������������������������������������������������������  151 Yumi Aoyama and Tetsuo Shiohara 11 Workplace Risk Assessment in Occupational Allergology ������������������  171 Dragan Mijakoski and Sasho Stoleski 12 Pesticide and Immunotoxicology������������������������������������������������������������  183 Tomoki Fukuyama and Risako Tajiki-Nishino 13 Clinical Evaluation of Plasma Decoy Receptor 3 Levels in Silicosis����������������������������������������������������������������������������������  197 Suni Lee, Shoko Yamamoto, Hiroaki Hayashi, Hidenori Matsuzaki, Naoko Kumagai-Takei, Tamayo Hatayama, Min Yu, Kei Yoshitome, Masayasu Kusaka, Yasumitsu Nishimura, and Takemi Otsuki 14 Reduction of Antitumor Immunity Caused by Asbestos Exposure����  215 Naoko Kumagai-Takei, Suni Lee, Hidenori Matsuzaki, Megumi Maeda, Nagisa Sada, Min Yu, Kei Yoshitome, Yasumitsu Nishimura, and Takemi Otsuki

Chapter 1

Mechanisms of Action of Inhaled Particulates on Allergic Lung Inflammation Etsushi Kuroda

Abstract  The incidence of allergic diseases is on the rise, especially in developed countries, and airborne particulate pollution from fine particulate matter and sand dust has been suggested as a factor in the exacerbation of allergic responses. These particulates function as adjuvants to induce allergic responses such as immunoglobulin E induction and eosinophil activation. This chapter summarizes data on the mechanisms by which particulates stimulate immune responses in the lung, including alveolar macrophage function and interleukin 1α release. Keywords  Particulate · Inflammasome · Immunoglobulin · Lymphoid tissue · Interleukin · Adjuvant · Alum · Apoptosis

Abbreviations DAMP damage-associated molecular pattern iBALT inducible bronchus-associated lymphoid tissue IgE   immunoglobulin E IL      interleukin MyD88 myeloid differentiation primary response 88 NLRP nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing OVA  ovalbumin E. Kuroda (*) Department of Immunology, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan Center for Vaccine and Adjuvant Research (CVAR), National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN), Osaka, Japan e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2020 T. Otsuki et al. (eds.), Allergy and Immunotoxicology in Occupational Health - The Next Step, Current Topics in Environmental Health and Preventive Medicine, https://doi.org/10.1007/978-981-15-4735-5_1

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PAMP pathogen-associated molecular pattern PM2.5 fine particulate matter with a diameter of 2.5 microns PRR    pattern-recognition receptor Tfh   T follicular helper Th2   type 2 T helper

1.1  Introduction The rising incidence of allergic diseases has become a severe public health problem, especially in developed countries, and epidemiological studies have reported that airborne particulate matter is associated with increased risk of hospitalization for asthma [1–3]. Typical allergic reactions are mediated by the induction of immunoglobulin E (IgE) responses and the activation of eosinophils; these are allergen-­specific immune responses that involve type 2 T helper (Th2) cells [4]. Since these Th2 cells are maintained as memory cells in allergic subjects, allergen-specific immune responses are evoked by reexposure to the identical allergen even if individuals remain in an allergen-free environment for a long period of time [5]. In general, the induction of memory cells, termed acquired immunity, is known to require the activation of innate immune responses [6, 7]. However, the mechanisms by which allergens stimulate innate immune cells and induce Th2 responses are unclear. Multiple studies have suggested that the induction of allergen-specific Th2 cells is promoted by innate immune cells activated by an adjuvant-like substance in the environment, such as fine particulate matter with a diameter of 2.5 microns (PM2.5) or sand dust [8–14]. Some particulates are known to exert strong adjuvant activity and induce Th2 responses [15]. This chapter introduces the putative immunological mechanisms of action of particulates and their role in lung immune responses.

1.2  Adjuvants and Innate Immune Responses Adjuvants are substances that induce or enhance antigen-specific immune responses. Some vaccines contain adjuvants that function as effective inducers of antigen-­ specific acquired immune responses, such as antigen-specific antibodies and cytotoxic T cells [16–18]. Activation of innate immune responses is necessary for the induction of acquired responses, and many adjuvants are known to activate innate immune cells. In general, innate immune cells are activated through the stimulation of pattern recognition receptors (PRRs) by pathogen-associated molecular patterns (PAMPs) such as pathogen-derived lipopolysaccharide or nucleic acid [19–22]. In addition, some factors from dying or stressed cells, termed damage-associated

Fig. 1.1  Adjuvant activity of particulates enhances allergen-specific antibody responses. Example from a study in mice sensitized by mite antigen in the presence or absence of particulate alum. Serum levels of immunoglobulin (Ig) G1 were quantified

Mite antigen-specific IgG1 (titer)

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Mite antigen

Mite antigen + particulate (alum)

molecular patterns (DAMPs), can also activate innate immune cells [23–25]. Thus, PAMPs and DAMPs are thought to function as adjuvants and induce acquired immune responses. Particulate matter such as PM2.5 and sand dust has been reported to function as an adjuvant [15], and to preferentially activate Th2 responses characterized by the induction of IgE and activation of eosinophils. Thus, some airborne particulates may be involved in the recent increase in the incidence of allergic diseases. In mice, low-dose exposures to allergens do not induce allergen-specific IgE, but administration of an allergen concomitantly with particulate aluminum salt (alum) as an adjuvant induces allergen-specific antibody (IgG1 and IgE) responses, indicating that particulates trigger and exacerbate allergic inflammation (Fig. 1.1). Unlike PAMPs, these particulates are not thought to stimulate PRRs on innate immune cells directly. Therefore, understanding their mode of action is key to identifying the immunological mechanisms underlying particulate-induced allergic inflammation.

1.3  Particulate and Adjuvant Effect Alum has been used as an adjuvant in human vaccines and its immunological action is well-characterized and known as the “depot effect.” Glenny et al. first noted that antigens adsorbed to alum exhibited persistence and prolonged release at the injection site [26], suggesting that alum is a long-term and effective stimulator of immune cell action [27]. However, alum adjuvanticity was shown to be normal even when alum nodules were removed several weeks after immunization [28], and ablation of the injection site after immunization with antigen/alum has been reported to not alter the magnitude of the immune responses [29], implying that the antigen depot is not necessary for the adjuvanticity of alum and other particulate adjuvants.

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Particulate adjuvants such as alum and silica were shown to stimulate innate cells (macrophages and dendritic cells) to activate the inflammasome, an intracellular complex of PRRs [30–32]. Particulate adjuvants mainly activate the nucleotidebinding oligomerization domain, leucine-rich repeat and pyrin domain-­containing (NLRP) 3 inflammasome, and then release interleukin (IL)-1β and IL-18 from innate cells via the action of caspase-1. Activation of the NLRP3 inflammasome was at first thought to be involved in the adjuvant activity of particulates [33], but the role of the NLRP3 inflammasome and caspase-1-dependent cytokines was questioned in several studies using NLRP3-, caspase recruitment domain-, and caspase-­1deficient mice [34, 35], and the inflammasome’s involvement in the adjuvanticity of particulates is still unclear. Since particulates such as alum and silica have been reported to induce cell death in phagocytes following phagocytosis [36–38], cell death has also been hypothesized to affect the adjuvant activity of particulates. DNA and uric acid are released from dying cells and may induce immune responses. Uric acid is a purine catabolite, and forms crystals at saturated concentrations. Both uric acid and its crystal are well-known DAMPs and exert strong adjuvant activity [39–44]. However, the mechanisms by which uric acid and its crystal stimulate innate immune cells and induce acquired immunity are unknown. Host cell-derived DNA is also a DAMP and functions as an adjuvant [16, 45–48]. Marichal et  al. demonstrated that host DNA was released at the site of alum injection and induced IgG1 and IgE responses, while coadministration of DNase and alum significantly reduced adjuvant activity [45]. A recent study also indicated that DNA release from neutrophils recruited to the site of alum injection was involved in alum adjuvanticity [48]. DNA released by activated neutrophils forms fibers termed neutrophil extracellular traps, which can contribute to the adjuvant activity of particulates, although the detailed mechanisms of innate cell activation by DNA are unclear. In addition to DAMPs, recognition of dead cells by a molecule expressed on dendritic cells has been reported to be involved in alum adjuvanticity [49]. Administration of alum induced apoptosis in cells that display phosphatidylserine on their surfaces, and CD300a expressed on innate cells bound to phosphatidylserine to clear apoptotic cells. CD300a-deficient mice exhibited reduced levels of antigen-­specific IgE and lower Th2 responses after immunization with alum and the antigen. CD300a is mainly expressed on inflammatory dendritic cells, and these cells are recruited at the site of alum injection. Inflammatory dendritic cells induced Th2 cells, suggesting that recognition of dead cells by specialized dendritic cells is involved in induction of acquired immune responses by alum [49]. Prostaglandin, a lipid mediator released from macrophages, also participates in alum-triggered IgE responses [37]. Thus, cell death and released DAMPs play a pivotal role in activating innate cells and inducing adequate acquired immunity. Table 1.1 summarizes the modes of action of particulate adjuvants reported so far.

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Table 1.1  Modes of action of particulate adjuvants Adjuvanticity factor Depot effect

Mechanisms of action Antigen absorbed to alum persists to stimulate immune cells over time. The depot effect has been questioned NLRP3 Some particulates stimulate innate cells to activate the inflammasome NLRP3 inflammasome, releasing interleukin 1β and interleukin 18. The importance of the inflammasome in the adjuvanticity of particulates is unclear Uric acid and its Known factors in damage-associated pattern recognition crystal and show strong adjuvant activity. Detailed immunological mechanisms are unknown DNA Released from dying cells and functions as an adjuvant. DNA released from activated neutrophils also exhibits strong adjuvant activity Apoptotic cells Inflammatory dendritic cells are activated via the interaction of CD300a and apoptotic cells, preferentially inducing type 2 T helper cell responses Lipid mediators The lipid mediator prostaglandin is released from particulate-activated innate immune cells and induces the production of immunoglobulin E MyD88 Signaling depends on the administration route. MyD88 signaling signaling is required for immunoglobulin E responses when particulates are administered directly to the airway

References [28, 29] (not required) [33–35]

[39–44]

[45–48]

[49]

[37]

[50, 51] (not required) [38, 52] (required)

1.4  Particulates and Lung Diseases Asbestosis and silicosis are representative lung diseases caused by inhalation of chemical particulates. The effect of silica in particular on the immune system has been demonstrated in animal models [53–56]. Inhaled particulates that are deposited in the lungs are generally engulfed by phagocytes such as alveolar macrophages and then excreted [57, 58]. Engulfing particulates has been reported to activate the inflammasome and subsequently IL-1β -via the action of caspase-1 [30–32, 59, 60]. In the lung model of inflammation caused by silica, inflammasome deficiencies have been shown to attenuate the magnitude of inflammation [55]. Exposure to tobacco smoke is a rodent model for chronic obstructive pulmonary disease, as tobacco smoke contains fine particulate matter, and long-term exposure has been shown to cause lung inflammation and the formation of ectopic lung lymphoid tissue. These responses are mediated by IL-1 and myeloid differentiation primary response 88 (MyD88) signaling [61–64]. Environmental particulates such as sand dust, diesel exhaust particles, and PM2.5 and industrial particulates such as carbon nanotubes have been assessed for their toxicity and allergenicity [8–14, 65–75]. Although multiple studies reported that these particulates also function as adjuvants to promote Th2-type immune responses similar to those induced by alum and silica,

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the underlying mechanisms are unclear [15]. Inhalation of particulates causes chronic lung inflammation and allergic asthma, so understanding the immunological mechanisms of particulate-induced inflammation would be required for prevention and effective treatment of chronic lung diseases.

1.5  Particulates and Alveolar Macrophages Alveolar macrophages act as sentinels against inhaled foreign substances [53, 76]. Some particulates, such as PM2.5, are deposited deep in the lungs. These particulates are thought to be engulfed by alveolar macrophages and cleared from the respiratory system [57, 58]. An in vitro study in the author’s laboratory used murine alveolar macrophages exposed to either inflammatory (alum, silica, and nickel oxide nanoparticles) or noninflammatory (aluminum oxide and hydroxyapatite) particulates. Inflammatory particulates stimulated alveolar macrophages to release IL-1α, which was closely linked to cell death caused by phagocytosis of the inflammatory particulates [38]. IL-1α has been reported to be constitutively stored in alveolar macrophages and rapidly released as a DAMP following cell death [53]; alveolar macrophage death therefore appears to trigger lung inflammation by releasing IL-1α [77], and was detected in  vivo in bronchoalveolar lavage fluid. Noninflammatory particulates did not induce macrophage death or IL-1α release in vitro or in vivo [38]. Multiple studies have reported that particulates activate the NLRP3 inflammasome after engulfment by dendritic cells and macrophages and then induce the release of IL-1β via caspase-1. Stimulation by low concentrations of lipopolysaccharide is known to be required for IL-1β release following activation of the NLRP3 inflammasome [30–32, 36, 59, 60], but this priming step is not necessary for IL-1α release from alveolar macrophages. IL-1β release was not detected in non-primed alveolar macrophages stimulated with alum. Unlike IL-1α, pro-IL-1β has no biological activity, and is therefore not considered a DAMP cytokine [77–80].

1.6  IL-1α Release and IgE Responses in the Lungs IL-1 is known to function as an adjuvant and induce antigen-specific IgE [38, 81, 82]. The author’s laboratory therefore studied whether IL-1α release from dying alveolar macrophages in response to particulate exposure was related to IgE.  Particulates were administered to mice by intratracheal instillation, followed by exposure to the allergen ovalbumin (OVA). Levels of OVA-specific IgE increased but only following administration of inflammatory particulates such as alum and silica. Mice deficient in the IL-1 receptor or IL-1α exhibited reduced levels of OVA-­ specific IgE,

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indicating that inflammatory particulates induce the release of intracellular IL-1α, which functions as an adjuvant to induce antigen-specific IgE responses [38]. IL-1-dependent IgE responses were observed in mice following inhalation of peanut flour, and serum IgE levels decreased significantly in IL-1 receptor-deficient mice, in accordance with the decreased numbers of T follicular helper (Tfh) cells [83]. Tfh-derived but not Th2-derived IL-4 has been shown to be necessary for class switching to IgE in B cells [84], suggesting that IL-1α participates in IgE responses via the differentiation of IL-4-producing Tfh cells. Activation of inflammasomes and IL-1β production do not appear to be involved in IgE responses caused by inhaled particulates [38].

1.7  Particulates and Unique Immune Responses in the Lungs Several studies have reported that MyD88 signaling is not required for the adjuvanticity of alum when it is administered intraperitoneally or subcutaneously [50, 51]. However, IL-1 receptor signaling is necessary for IgE responses when alum is administered by intratracheal instillation, suggesting that MyD88 signaling is involved in the adjuvanticity of alum in the lungs. Matsushita and Yoshimoto also reported that MyD88 signaling was necessary for IgE responses by intranasal administration of alum [52]. These data suggest that direct administration of inflammatory particulates to the airway induces IgE responses via mechanisms specific to the administration route. As mentioned above, alveolar macrophages constitutively express intracellular IL-1α, and release it as a dead cell factor induced by phagocytosis of inflammatory particulates. This is unique to alveolar macrophages and not observed in macrophages from other tissues stimulated with inflammatory particulates [38, 53]. Lung tissues from mice exposed to particulate alum and OVA exhibited infiltrations of inflammatory cells and lymphoid cluster formations. These lymphoid clusters were mainly composed of B cells and hypothesized to be inducible bronchus-associated lymphoid tissue (iBALT) [38]. iBALT is known to be induced in chronic inflammation evoked by viral infection, administration of lipopolysaccharide, or autoimmune diseases such as rheumatoid arthritis [85–90]. iBALT formation was also observed in the lungs of mice with allergic inflammation [91, 92], and pathogenic Th2 cells have been associated with iBALT formation [5, 93], suggesting that lung ectopic lymphoid tissues are closely linked to allergic inflammation. The lymphoid clusters induced by alum and antigen exposure contain a germinal center of B cell maturation and antibody class-switching, areas of T cells, and clusters of plasmablasts. These iBALT formations are regulated by IL-1 and are correlated with IgE levels [38]. iBALT structures are involved in  local lung immune responses and dendritic cells recruited to the lungs induce and maintain the iBALT structures [87]. Mice deficient in the IL-1 receptor have decreased numbers of iBALT formations and

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recruited dendritic cells in their lungs [38, 94]. This suggests that dead cell-derived IL-1α is involved in dendritic cell infiltration and subsequent formation of iBALT structures, inducing IgE responses. Both iBALT structure formation and IgE induction have been shown to be reduced following depletion of lung dendritic cells [38]. Furthermore, Tfh cells have also been reported to participate in the formation of iBALT structures [89], and particulate-induced iBALT formations were reduced in Tfh cell-deficient mice, mirroring antibody responses [38]. Dead cell-derived IL-1α may therefore induce the activation of both Tfh cells and dendritic cells, resulting in iBALT formation and IgE responses as immune responses unique to the lung. Figure  1.2 summarizes the model of particulate (alum)-induced allergic lung inflammation.

Inhalation of particulates

Inflammatory particulates

Non-inflammatory particulates

Engulfment of particulates

No cell death

iBALT formation Cell death and IL-1α release Allergic inflammation

No inflammatory responses

Fig. 1.2  Model of particulate-induced lung allergic inflammation. Inhaled inflammatory particulates kill alveolar macrophages and induce the release of interleukin (IL)-1α, which functions as an adjuvant to promote the formation of inducible bronchus-associated lymphoid tissue (iBALT) and production of immunoglobulin E. Modified figure from Kuroda et al. [38]

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1.8  T  herapeutic Strategies for Particulate-Induced Lung Inflammation iBALT structures in the lungs have been observed in infants but not in healthy adults [95, 96], indicating that infants are sensitive to particulate-induced allergic asthma. Apart from environmental particulates, tobacco smoke constituents can also induce iBALT structures in the lungs, suggesting a similar inflammatory mechanism [63, 64]. Targeting IL-1 signaling may therefore be a valid therapeutic strategy for particulate-­induced lung inflammation. Corticosteroids are generally used to treat allergic asthma, and have been demonstrated to hamper the maturation of iBALTs induced after particulate and antigen exposure [97]. Administration of corticosteroids may control allergic asthma in part by inhibiting iBALT formation.

1.9  Conclusion This chapter summarized the mechanisms of allergic lung inflammation caused by inhalation of particulates. Allergic lung inflammation diseases are frequently observed in urban areas of developed and fast-growing countries, and particulate pollution is hypothesized to be their major trigger. However, not all particulates function as adjuvants to induce inflammation, and some do not activate immune responses in the lungs following direct administration to the airway. These particulates have no effect on macrophage function and its subsequent IL-1α release. Effective treatment and prevention of inflammatory diseases caused by inhaling particulates require the elucidation of the immunological mechanisms involved.

Funding  The author received a Grant-in-Aid for Scientific Research from the Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT) and the Japan Society for the Promotion of Science (JSPS) (MEXT/JSPS KAKENHI grant numbers JP24591145 and JP16H05256), the Research on Development of New Drugs from the Japan Agency for Medical Research and development (AMED) (grant number 18ak0101068h0002), and the Japan Science and Technology Agency (JST) PRESTO (grant number JPMJPR17H4).

References 1. Kanatani KT, Ito I, Al-Delaimy WK, Adachi Y, Mathews WC, Ramsdell JW, et al. Desert dust exposure is associated with increased risk of asthma hospitalization in children. Am J Respir Crit Care Med. 2010;182(12):1475–81. https://doi.org/10.1164/rccm.201002-0296OC.

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Chapter 2

Metal Nanoparticle Health Risk Assessment Luca Di Giampaolo, Claudia Petrarca, Rocco Mangifesta, Cosima Schiavone, Cinzia Pini, Alice Malandra, Francesca Bramante, Alessio Pollutri, Michele Di Frischia, and Mario Di Gioacchino

Abstract  The widespread application of nanomaterials confers enormous potential for human exposure and environmental release particularly for workers producing nanoparticles or making nano-based objects. The various routes by which nanoparticles could be taken up by the body (respiratory, skin, and digestive) complicate the definition of NPs to be used in risk assessment. The present review describes the difficulties in making a sufficiently correct risk assessment and management for nanomaterials, addressing the various problems that render difficult the risk management of nanomaterials in the occupational setting, in particular the exposure scenario, the exposure appraisal, and the hazard identification and characterization.

L. Di Giampaolo School of Specialisation in Occupational Medicine, “G. D’Annunzio” University, Chieti, Italy Unit of Immunotoxicology and Allergy, Department of Medicine and Aging Sciences (DMSI) and CAST, University G. d’Annunzio of Chieti-Pescara, Chieti, Italy C. Petrarca · R. Mangifesta · C. Schiavone Unit of Immunotoxicology and Allergy, Department of Medicine and Aging Sciences (DMSI) and CAST, University G. d’Annunzio of Chieti-Pescara, Chieti, Italy C. Pini · A. Malandra Unit of Allergy, University Hospital, Chieti, Italy F. Bramante · A. Pollutri · M. Di Frischia School of Specialisation in Occupational Medicine, “G. D’Annunzio” University, Chieti, Italy M. Di Gioacchino (*) School of Specialisation in Occupational Medicine, “G. D’Annunzio” University, Chieti, Italy Unit of Immunotoxicology and Allergy, Department of Medicine and Aging Sciences (DMSI) and CAST, University G. d’Annunzio of Chieti-Pescara, Chieti, Italy Unit of Allergy, University Hospital, Chieti, Italy e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2020 T. Otsuki et al. (eds.), Allergy and Immunotoxicology in Occupational Health - The Next Step, Current Topics in Environmental Health and Preventive Medicine, https://doi.org/10.1007/978-981-15-4735-5_2

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Keywords  Nanoparticles · Environmental monitoring · Exposure appraisal · Risk assessment · Risk management · Control banding

2.1  Introduction Nanoparticles are defined as “a material with one, two, or three external dimensions at the nanoscale (from ~ 1 to 100 nm) metrics.” In this form, they achieve unique mechanical, optical, electrical, and magnetic properties. Recent rapid advancements in nanotechnology have led to wide applications of nanomaterials in a number of areas, including material science, energy, and medicine. Therefore, the widespread application of nanomaterials confers enormous potential for human exposure and environmental release particularly for workers producing nanoparticles or making nano-based objects. The wide variety of routes by which NPs could be taken up by the body (respiratory, skin, and digestive) complicates the definition of NPs to be used in risk assessment. It is probably necessary to consider multicomponent and multiphase particles of any size and composition that can be absorbed by the body. Although the potential for these nanomaterials to affect the ecosystem function, to exert cytotoxic and pro-inflammatory effects in vitro as well as to induce early alterations in different target organs in vivo models has been reported, further investigations appear absolutely necessary to confirm such preliminary findings [1]. Depending on the conditions of manufacture, formulation, use, and final disposal, a risk assessment of NPs may need addressing: (1) Worker safety: typically workers are exposed to higher levels of chemicals and for more prolonged periods of time compared with the general population; (2) Safety of consumers using products that contain NPs; (3) Safety of local human populations due to chronic or acute release of NPs from industry; (4) The potential for human reexposure through the environment. Focusing on products that are deliberately used in nanoparticle form in the environment, such as biocides, or environment-improving agents; (5) The environmental and human health risks involved in the disposal or recycling of nanoparticle-­based products. In principle, the traditional risk assessment procedure is an appropriate tool for assessing the risks from exposure to NPs under specified exposure conditions. The traditional risk assessment methodology comprises the following stages: (a) exposure appraisal; (b) hazard identification and characterization; and (c) risk characterization and management. This framework has not yet been applied to NPs, in terms of either their potential human or environmental impact. There is an unclear situation with regard to regulatory requirements for risk assessment. As a consequence, there are no official guidelines on what constitutes an appropriate testing regimen. This review will focus on metal nanoparticles due to the large and already increasing use of these nanoparticles in industries.

2.2  Exposure Scenarios Essentially workers are exposed to NP by inhalation, ingestion, and contact. In a room, the air can contain 10,000–20,000 NPs/cm3, in a street the quantity on NPs are extremely higher reaching 100,000 NPs/cm3. In a working environment, their

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concentration is extremely variable, according to the type of working activity and the hygienic measures applied [2]. Therefore, workers can be in contact with millions on NPs per hour. It is estimated that half of them can reach the alveoli or are in contact with the skin. A part of them are ingested and adsorbed through the gastrointestinal system. NPs are adsorbed and can be found in tissues, penetrate cells (through diffusion or endocytosis), and exert their toxic potential. It has been observed that NPs may trigger oxidative stress, inflammation, and indirect DNA damage in living systems, and that the size and shape of NPs could have an important role in determining the cellular damage. In many cases the toxicity of NPs is exerted by released ions [3]. In some cases, the released ions are neutralized by forcing or interacting with the cell component leading to depletion of dissolved oxygen and generating reactive oxygen species (ROS) together with reactive nitrogen species (RNS) leading to molecular and biochemical alterations [4].

2.2.1  Lung Exposure The respiratory tract is the most important route of exposure to NPs. Their deposition in the alveolar region gives the possibility of their absorption in the lung. Although NPs tend to agglomerate, potentially making their aerodynamic characteristics similar to those of larger particles [5], size remains a characteristic, which correlates with toxic responses. However, even though surface area has been completely studied for inflammatory responses, it has not been similarly validated for cytotoxicity or oxidative stress effects [6]. NPs, deposited in the respiratory tract, easily translocate from the alveolar region to epithelial and interstitial sites [7]. Muhlfeld [8] demonstrated that when NPs reach the alveoli, they have a high probability of encountering the alveolar epithelium, because the uptake of NPs by alveolar macrophages seems to play a minor role than for larger sized particles [9, 10]. Essentially monocytes/macrophages transport NP across a confluent endothelial cell layer [11]. NPs enter easily interstitial spaces after alveolar deposition, compared with bulk particles [12]. Inhaled NPs can spread more like gas molecules and, thanks to their size, NPs pass through the alveoli into the bloodstream, reaching sensitive sites such as bone marrow, liver, kidneys, spleen, and heart [8]  and specifically accumulate at sites of vascular disease [13]. Videira et al. [14] evidenced inhaled 200 nm solid lipid NPs (SLNPs) rediolabeled with 95Tc into the lymphatics, and a high rate of distribution in periaortic, axillar, and inguinal lymph nodes. It has been showed that, in the blood of rats exposed to 15 nm Ag NPs by inhalation, or to agglomerated Ag NPs or Ag+ ions intratracheally, the significant amounts of silver detected initially decrease rapidly, and this shows that systemic distribution occurred [15]. Hint of silver were found in the liver, kidney, spleen, brain, and heart, while the nasal cavities, such as the posterior portion, and lung-associated lymph nodes showed relatively high concentrations of silver. Exposure via inhalation by rats of 40 and 51 nm CdO resulted in efficient deposition in the lung [16], with a fraction from the lung translocated to

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the blood, liver, and kidneys. Kwon et  al. [17] studied the body distribution of inhaled 50 nm fluorescent magnetic NPs in mice. Magnetic resonance imaging and confocal laser scanning microscope analysis demonstrated that NPs were distributed in various organs, for example, the liver, testis, spleen, lung, and brain, indicating that these NPs could penetrate the blood–brain barrier. Dumková [18] showed that sub-chronic inhalation of lead oxide nanoparticles favors their broad distribution and tissue-specific subcellular localization in target organs. Thanks to a single 4-h nose-only exposure to freshly emitted or aged CeO2 in different gaps of time, ICP-MS detection of Ce in the lungs, gastrointestinal tract, spleen, kidneys, heart, brain, liver, blood, olfactory bulb, urine, and feces were studied to have a complete vision of the distribution. Their research found Cerium mostly in the lungs and feces, with extrapulmonary organs’ contributing less than 4% to the recovery rate at 24 h post exposure. No significant differences were found in biodistribution patterns between fresh and aged CeO2 nanoparticles [19]. Oberdorster et al. [20] showed that after inhalation of (20–29 nm) 13C NPs, a transportation from the olfactory mucosa of the rat to the olfactory bulb occurred, supplying a portal of entry into the central nervous system (CNS) for solid NPs. The same group of the former study [21] performed experiments in rats inhaled with 36 nm 13C NPs. During inhalation about 20% of the particles deposited into the nasopharyngeal region reached the olfactory mucosa and further translocated to the olfactory bulb where they persisted. The NPs were also able to cross the blood– brain barrier in some regions, targeting the CNS. The translocation of NPs to the brain was also found in mice exposed by inhalation to 20–200 nm TiO2 NPs [22], gold NPs [23], and Ag NPs [24]. We can assert that it is mostly accepted that the translocation of NPs from lung to other tissues can occur. Nevertheless, only a small part of inhaled NPs translocates across the air–blood barrier, entering the circulation and reaching other organs. To understand the significance of this translocation, research priorities should be: (1) to study the metabolic fate of the translocated NPs; (2) to assess their cardiovascular toxicity: cardiovascular system is currently considered an important potential target for NPs; (3) to study the toxic effects of the translocated NPs with the help of in vitro systems such as perfused organs and cell cultures; effects investigated would involve systems which are particularly sensitive to long-term low-dose exposure, such as the CNS and reproductive system; and (4) to study mechanisms of NP penetration into cells and their intracellular distribution in relation to the immunotoxic responses and other effects, particularly the production of reactive oxygen species (ROS) and inflammation proteins.

2.2.2  Dermal Exposure Many studies have suggested that metallic NPs can trigger sensitization reactions. However, there is an urgent need to clarify the immunologic effects of skin exposure to metallic NPs, especially in how they aggravate allergies. Although many studies

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have been conducted to explore toxicity following skin exposure to metallic NPs, there is a lack of knowledge of immunologic mechanisms. In general, haptens are involved in immunoreactivity, but are not immunogenic. They have the potential to modify self-protein binding to obtain immunogenicity. In contrast to classic haptens, transition metals produce coordination complexes instead of stable covalent modifications with binding proteins. These coordination complexes are reversible and exchange allergenic metallic ions among different sites [25]. When foreign substances enter the body, immune cells, such as antigen-­presenting cells and leukocytes, recognize them and activate immunodefenses. Smulders et al [26]. showed an increased Ti concentration in draining lymph-node cells after topical application of TiO2 NPs, indicating that TiO2 NPs penetrated the skin and were transferred to the lymph nodes. Human macrophages were found partially to dissolve ZnO NPs based on evaluations of ZnO NP counts with X-ray fluorescence and SEM [27]. Therefore, one of the potential immunologic mechanisms after metallic NPs are topically applied might be that the NPs move to the lymph nodes, where they are engulfed by macrophages. On the other hand, metallic NPs activate adaptive immune responses. Metallic ions, such as Ni ions, were capable of activating metallic ion-specific CD4+ T cells in the lymph nodes, and IL17 was produced in response to metallic ions. On the basis of these studies, metallic NPs have been speculated to act as carriers to transport metallic ions into the lymph nodes for CD4+ T cell- and IL17-mediated immune responses. In the case of inflammatory skin diseases, such as atopic dermatitis and psoriasis, the exact immune responses and whether these responses can affect skin inflammation are not yet fully understood. Increasing skin exposure of metallic NPs from a variety of nanotechnology applications has raised concerns regarding potential adverse effects on human health. Of all possible entry routes, skin absorption may serve as the first portal for metallic NP exposure. As mentioned, transdermally applied metallic NPs can penetrate damaged or even intact skin. The high activity of metallic NPs has raised debate on their interactions with skin cells. Although some studies have been conducted to assess these interactions, their results were inconclusive. The proposed mechanism is that metallic NPs generate oxidative stress, mitochondrial damage, and DNA damage, and accelerate the apoptosis of skin cells [28] and have synergistic biological effects [29]. The effects of Fe3O4 and ZnO NPs on mouse dermal fibroblast cells have been evaluated in vitro. After exposure, a number of endocytic vesicles were detected on the cell membrane, and metallic NPs were visualized in either the cytoplasm or cytoplasmic vesicles, indicating that metallic NPs seemed to be phagocytosed by cells. In addition, NP-treated cells formed irregular shapes due to cytoplasmic shrinkage or were even completely necrotic at high NP concentrations [30]. Furthermore, NP attachment caused membrane rupture, increased cell-membrane permeability and influenced cell-membrane fluidity [31]. Moreover, the combination of electrostatic attraction and hydrogen bonding between NPs and membranes was probably one of the reasons for membrane disruption and gelation [32]. Nowadays, we do not have sufficient conclusions to understand the specific mechanisms of transdermally applied metallic NPs. Actually, there is still debate as

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to whether metallic NPs can penetrate the normal skin barrier. Additionally, the interactions of metallic NPs and skin cells remain controversial. These inconsistent results may be caused by several factors, such as different types of NPs and physicochemical characterizations, varying experimental approaches, and various cellular or animal models used in the experiments [33].

2.2.3  Gastrointestinal Exposure The ingestion of NPs can happen through contaminated foods or swallowing saliva or nasal fluids contaminated with environmental NPs. When nanoparticles have entered the buccal mucosa, they can impact on the physiological homeostasis of the human body in different ways. First of all, nanoparticles can directly strike the buccal epithelium. It was already showed that nano-TiO2 particles have the potential to generate ROS and induce oxidative stress [34], which usually results in inflammation and/or cell death [35]. Second, free particles may pervade the epithelium and enter systemic circulation. It was demonstrated that nanoparticles accumulated in liver, spleen, lung, and kidney after oral administration [36]. NPs can permeate the digestive tract by direct ingestion [37], and dental prosthesis debris [38]. Nanoparticles can be easily absorbed by the digestive tract not only through the M-cells in the Peyer’s patches and the isolated follicles of the intestinal associated lymphoid tissue, but also by transcytosis via enterocytes [39]. The primary components of the GIT include the mouth, esophagus, stomach, small intestine (duodenum, jejunum, ileum), and large intestine (cecum, ascending colon, transverse colon, descending colon, sigmoid colon, and rectum). The small intestine is the site of most nutrients digestion and this is the spot where absorption into the bloodstream of NPs would occur, especially in the segments of the jejunum and ileum [40]. The present available studies about the degree of TiO2 particle uptake and absorption from the GIT into the blood circulation are not consistent, and may be species dependent, as studies in mice appear to differ from reported findings with TiO2 exposures in rats and humans [41–44]. Other factors include the type of NPs, as well as important physicochemical characteristics including particle size, dispersibility, and charge. In general, the majority of biokinetic studies show that most of the ingested TiO2 NPs are not absorbed into the bloodstream but instead are excreted from the GIT. It is important to think about what happens to NPs contained in foods: will they remain in the intestine or will they move on into the body? The intestine should take up nutrition and protect the body from unwanted substances in the food. It is not known whether NPs are regarded as “unwanted substances” and excreted or not. The hypothesis that nanomaterials will not remain there for indefinite periods is led by the rapid transit of material through the intestinal tract (on the order of hours), together with the continuous renewal of epithelium. The extent of particle absorption in the gastrointestinal (GI) tract is affected by size, surface chemistry and charge, length of administration, and dose [45].

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Once ingested, NPs enter the stomach and are submitted to usual digestive processes that may afflict them. Meng et al. [46] have demonstrated that 23.5 nm Cu NPs consume the hydrogen ions in the stomach more quickly than microparticles, converting the Cu NPs into cupric ions whose toxicity is very high. NPs surviving on the gastric digestion can be absorbed in the enteric tract. A consistent absorption was observed, with a systemic distribution to liver, spleen, blood, and bone marrow. Particles larger than 100 nm did not reach the bone marrow, and those larger than 300 nm were absent from blood. No particles were detected in heart or lung tissue. The uptake was mainly via M-cells (specialized phagocytic enterocytes) of the Peyer’s patches, with translocation into the mesenteric lymph and then to systemic organs. A further possibility for intestinal uptake of NPs is via enterocytes [45]. In a study performed in mice orally administered with 4 and 58 nm gold NPs, Hillyer and Albrecht [47]  showed the capture of gold NPs by the intestine, their passage through the blood and their translocation to the brain, lungs, heart, kidneys, intestine, stomach, liver, and spleen. The uptake occurred by persorption through holes created by extruding enterocytes. This effect was inversely proportional to the size of the NPs: the smaller the particle, the greater was the passage.

2.2.4  Interaction of NPs with Immune System NPs can interact with human immune system once they enter in the body [48]. Effects of NPs on this system can be of various types, in a range that spreads from a specific immune response to immunosuppression and autoimmunity, based mainly on size and chemical properties of NPs [49–52]. Effects can be also of indirect types due to NPs’ capacity to change ionic homeostasis of a system. Currently there are no information in scientific literature of diseases caused by NPs, with only exception of contact dermatitis due to Pd NPs. Most studies were focused on effects of metal NPs (MeNPs) on immune system, and it was seen that innate immune system cells react to MeNPs in a similar pattern of how they react to pathogen microorganisms. Nanotubes administered intratracheally were found to produce dose-­dependent lung lesions differently from carbon black [53], and multifocal pulmonary granuloma, effects different from those of quartz, carbon black, and graphite [54]. In some studies, it was demonstrated that subtoxic concentrations of ZnO NPs downregulated CD16 expression on NK-cells or AuNPs inhibited TLR-9 function in macrophages. In vitro, exposition of macrophages to MeNPs caused pro-­ inflammatory effects with increase of productions of various cytokines including IL8 and HSP70, mainly small NPs (< 5 nm of diameter). This effect was confirmed on human blood monocytes. CoNPs induced in peripheral blood lymphocytes and monocytes an increase in production of TNF-α and INF-γ and inhibition in production of IL-10 and IL-2 [55]. CoNPs in particular showed the capacity of activating  immune system more strongly than other MeNPs. Two studies demonstrated that CoNPs have lower cytotoxicity than microparticles and ions, and also showed

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that only CoNPs and microparticles had morphologic transforming potential [50, 56, 57]. Furthermore, Pd-NPs induce PBMC release of cytokines different from those released in presence of Pd ions [58, 59]. In conclusion it is clear that NPs can interact with immune system and can be relevant to increase in many diseases that in Western countries are increasing in number and social costs (allergies, autoimmunity, and cancers) despite today in literature no disease was clearly associated with NPs (except for allergic contact dermatitis). NPs can have a role in allergic sensitization because they can act like haptens and can elicit a Th2 response in lymphocyte population with the production of IgE antibodies by B cells [60]. In addition to direct sensibilization, NPs have the capacity to show also an adjuvant effect that may favor allergen sensitization. It was seen that the size of NPs may change the type of immune system response, with big NPs (>100 nm) more inclined to elicit a Th2 response, while little NPs (~50 nm) more inclined to elicit a Th1 response. Even chemical composition, dose, shape, and time of exposure can influence the types of responses to NPs [60].

2.3  Exposure Appraisal The exposure appraisal should answer six primary questions: (1) How, when, and where does exposure occur? (2) Who is exposed? (3) How much exposure occurs? (4) How does exposure vary? (5) How uncertain are exposure estimates? and (6) What is the likelihood that exposure will occur? The greatest exposure to NPs pertains to workers during production and transfer of the intermediate or final product to other handling steps. Exposure of the public to NPs can only occur through the product or release of NPs to the environment. Nevertheless, there does not exist to our knowledge a systematic approach related to the control of NP production and products. During a 2004 workshop in Brussels, experts of the European Commission suggested the development of a nomenclature for intermediate and finished engineered nanomaterials, assigning a universally recognized Chemical Abstract Service (CAS) Number to engineered NPs [61]. In particular some factors have to be studied and discussed to measure the exposure potential. Relevant factors are the extent of exposure (time and concentration), the uptake route (inhalation, transdermal, ingestion), and the probability of exposure. No systematic approach is currently available for measuring the probability of exposure related to NP production and handling processes. Oberdorster et al. [62] showed the substantial differences in mass concentrations and surface areas for particles: study showed that the use of mass concentration data alone is insufficient, and the number concentration and/or surface area need to be included. Sampling of NPs is a great challenge. The sampling strategy should ensure that the particle collection methods represent as accurately as possible the real exposure at the site in question, and methods should be developed according to the size and nature of the particles under investigation. The separation of NPs from larger particles can only be reached at a relatively high pressure drop. Technologies to measure

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some of these metrics for NPs in situ have been identified, for example to determine number concentration. However, these are not readily available, particularly in a form which may be used to measure personal exposure on a routine basis. The results of the measurements can confirm the suspected temporal and spatial variation in (number) concentration and aerosol size distribution. Anders et  al. [63] reported the important role of serum proteins in modifying the ZnO nanocrystal surface resulting in the formation of considerably smaller agglomerates and stable NP dispersions. Riediker et al. [64] concluded that there are major similarities about the effects that particles in wide size range can induce. Substantial differences can be seen in deposition, translocation, and clearance, especially for inhalation exposures. In a review, Boccuni [65] reported selected techniques that can evidence the presence or absence of nanomaterials along with their comprehensive exposure measurement, that allow the quantification of NPs in the workplace. Further researches are needed to allow accurate quantification of personal exposure of workers. To raise the accuracy of risk assessment we need the development that describes the dispersion and transformation of NP and their agglomerates in the working environment. It is also crucial to set strategies and standard measurement methods to harmonize exposure data for risk assessment and to enable the development of safety standards.

2.4  Hazard Identification and Characterization Health effect data on workers exposed to NPs are limited because of the incipient nature of the field, the relatively small number of workers potentially exposed to date for which the exposure has been sufficiently evaluated, and the lack of time for chronic disease to develop and be detected. Human data are derived from exposures to ultrafine and fine particles, which have been assessed in epidemiological air pollution studies and in studies of occupational cohorts exposed to mineral dusts, fibers, welding fumes, combustion products, and poorly soluble, low-toxicity particulates such as titanium dioxide and carbon black [35, 60, 66]. Many data, essentially related to exposure to engineered NPs, also are derived from animal studies [53, 54, 62, 67–71]. A strong positive correlation exists between the surface area, oxidative stress, and proinflammatory effects of NPs in the lung [35, 62]; however, the extrapolation of animal studies to humans needs a prudent evaluation. Although the findings are not conclusive, various studies of engineered NPs in animals raise concerns about the existence and severity of hazards posed to exposed workers [72]. Possible adverse effects include the development of fibrosis and other pulmonary effects after short-term exposure to carbon nanotubes [62, 70, 71], the translocation of NPs to the brain via the olfactory nerve, the ability of NPs to translocate into the circulation, and the potential for NPs to activate platelets and enhance vascular thrombosis [73].

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For poorly soluble, low-toxicity dusts such as titanium dioxide, smaller particles in the nanometer size range appear to cause an increase in risk for lung cancer in animals on the basis of particle size and surface area [62, 74–76]. There are few evidences in humans, as example in 2009 seven healthy women exposed to polyacrylate NPs in work place experienced pleural effusion and dyspnea, or a 22-year-old chemist who experienced throat congestion, flushing in the face, rhinitis, and erythema multiforme-like after exposition to dendrimers. Sneezing and contact allergic dermatitis were frequently reported in workers exposed to NPs [60]. None of these findings are conclusive about the nature and extent of the hazards, but they may be sufficient to support precautionary action. Ultimately, the significance of hazard information depends on the extent to which workers are exposed to the hazard [77]. It is evident that there are sufficient data in the literature to conclude that, from a risk assessment point of view and for some types of NP at least, it is not valid to rely entirely on toxicological findings from testing the component of an NP of interest in another physical form. An approach to hazard identification and characterization for a chemical of interest could be the following: (a) If there are considerable available data in the literature, the hazardous properties of its nanoforms should be evaluated in a test battery. It must be reiterated that it is not scientifically valid to rely exclusively on the properties of the chemical in other physical forms for risk assessment purposes; (b) If NPs have very similar hazard properties to other physical forms, further work on hazard assessment on the NPs may not be necessary; (c) If the NP form has substantially unique properties and no information is available on its biological properties, suitable exposure methods should be used to evaluate their toxicity. The question that needs to be addressed in this case is: what is the full package of tests that needs to be conducted? The selection of this test battery should be informed by knowledge of the chemical, physical, and biological properties, along with data on the same chemical in other physical forms. In vitro tests play an important role in this screening process; in principle, combined with information on the surface chemistry, these tests could provide an important early indicator of the differences or similarities in potential hazard between the NP form of a substance and other physicochemical forms. However, characterization of the uptake, distribution, deposition, and retention of NPs and the comparison with their larger sized counterparts may require an in vivo approach. An interesting study was made in 2016 to assess the risk of oral exposure to titanium dioxide particles, and in this study the authors tried to establish some methods to characterize the risk [78]. Authors used two different approaches: (1) Traditional approach based on external exposure in humans and in dose levels in animals determining no adverse effects (NOAEL) or determining adverse effects (LOAEL); (2) Internal dose approach based on assessing internal dose levels using toxicokinetic data. It considers the organ concentration of a dose at which adverse effects appear and tissue accumulation of a substance over time. The approaches to assess exposure have some critical issues, for example kinetic model used on internal dose approach is only partly mechanism-based, because

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knowledge about kinetic of TiO2 NPs is limited. Moreover, oral absorption is critical, because not all dose is absorbed and then redistributed to organs and no long-­ term studies are available today [78]. With traditional approach, TiO2 NPs did not show adverse effects for liver and spleen, but potential risks could not be excluded for testes and ovaries, while potential risks for spleen, liver, testes, and ovaries were present with internal dose approach. The difference between these two approaches shows the importance to consider the toxicokinetic in assessing the risk, especially in the case of substances able to accumulate in human tissues. This aspect is particularly relevant in case of accumulation of a substance in a scenario of a life-­ long exposure, in which it can reach considerable concentrations in various organs. In conclusion, screening assessments of exposures to the more studied NPs could be conducted by developing toxicity benchmarks using the weight of evidence from studies of: (a) nanoscale forms in the toxicological and pharmacological literature; (b) fine-scale forms corrected for the proportionally greater surface area of nanoscale particles; (c) more toxic particles such as UFP; and (d) the toxicology and epidemiology of metal fumes. Uncertainties in such assessments will have to be considered given data limitations; however, collectively, the available studies are beginning to reveal important features necessary for initial risk assessments of specific NPs.

2.5  Risk Characterization and Management Health effects and risk data on workers exposed to NPs are yet limited, despite some studies showing that exposure to NP may promote some adverse effects on organ, tissue, cellular, subcellular, and protein levels, as mentioned above. Moreover, the toxicity of chemicals, like NPs, depend on the dose, time of exposure, and route of administration. Risk assessment of nanomaterials is currently considered as a scientific challenge for stakeholders and there is an ongoing discussion on how to consider nanomaterials in well-proven risk assessment approaches which initially were developed for conventional chemicals. First requisite for evaluations of workers’ exposure requires a comprehension of potential hazards as well as a reasonable exposure estimate, but in many circumstances the exposure assessment cannot be quantified, due to technical limitations of measurement (methodologies) either in the workplace or in the environment, and often times the exposure is so low that it cannot be measured. Therefore, often times the exposure evaluations require estimations quantified by product life cycle [40]. Due to the lack of available data on the risk characterization of different NPs, no generic conclusions are possible at this stage. Consequently, each product and process that involves NPs must be considered separately in terms of worker safety during the manufacture of NPs, safety of consumers using products that contain NPs, safety of local populations due to chronic or acute release of NPs from manufacturing and/or processing facilities, potential human health risk for reexposure through the environment due to disposal or recycling of NP-dependent products.

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In the absence of suitable hazard data, a precautionary approach should be adopted. Containment and control of potential exposures should pursue protection practices relevant to the activity of worker and commensurate with the available control measures. The control measures include elimination of the substance, replacement with a safer substance, use of PPE, and control of procedures for the use of the substance. In addition, it should also be noted that there is no reliable information on the effect of the simultaneous exposure to multiple forms of NPs. It would be appropriate to assume that the effects are additive, or there could be interactions between NPs and other stressors (either physical, chemical, or biological) which should be considered on a case-by-case basis. The main source of information on the potential for adverse human health effects with NPs are the epidemiological and in vitro studies of airborne particles in ambient air. A wide variety of in vitro assays are available to assess cellular toxicity. In vitro assays are used in most studies  to evaluate the cytotoxicity and biological responses of NPs [79]. Researchers often tend to implement comparatively simple in  vitro test systems that are relatively easy to perform, control, and interpret. However, there is a need to develop validated in  vitro assay systems for toxicity testing of an expanding range of NPs. In vitro methods can be precisely controlled; hence, they can provide more reproducible toxicity data than in  vivo models but require higher standardization [80]. The studies have shown that smaller particles of low solubility (less than 1  μm) are substantially more toxic than larger particles. Furthermore, it has been found that as far as ambient air pollution with fine particles is concerned, there is a population subgroup (including individuals with severe chronic respiratory and heart disease) that is much more sensitive to the adverse effects than the public as a whole [81]. There is some evidence that NPs can have a different toxicity compared with larger particles of the same substance, for example, the different modulation of cytokine production by mononuclear cells exposed to CoCl2, microparticles, and NPs [55]. Co microparticles showed a greater inhibitory effect compared with other Co forms. Equally CoNPs administered intratracheally were found to produce dose-dependent lung lesions differently from carbon black [82], and multifocal pulmonary granuloma, effects different from those of quartz, carbon black, and graphite [54]. Furthermore, there are differences in toxicity even between NPs in molecular or ionic form as highlighted in in vitro experiments. For example there is a significant increase of intracellular ROS in Pd(IV) exposed human PBMC cells, but not in Pd-NPs exposed cells; moreover, cells exposed to Pd(IV) ions showed a significant amplification of cell cycle arrest in the G0/G1 phase and a significant reduction in the GS and G2/M phases [3]. The conclusion from these studies is that NPs have a specific activity/toxicity and support the case for a separate/additional risk assessment of substances that are in NPs form. These differences may be attributable to the fact that they have a much greater surface area to weight ratio than larger particles and, as a consequence, they tend to be more chemically reactive and bind other substances to their surface more effectively. Because of the inverse relationship between particle size and surface area, it is imperative that dose–effect (or concentration–effect) relationships are established as a function of total surface area and/or number of particles, rather than

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mass units. Furthermore, a comparison should be made between the effects of the conventional and the NP form, and further between NPs in molecular or ionic form. The International Organization for Standardisation (ISO) has established a number of technical reports to provide a framework for risk assessment of nanotechnologies such as ISO/TR12855 and ISO/TR13121. Risk management includes: (a) the identification of the hazard (describe NPs, their applications, physicochemical profiles, hazard profiles, and exposure profile); (b) the evaluation of the risk based on the combination of type of hazard, mode, and time of exposure and potential risks; (c) control risk with the possibility of intervening at various levels (level 1: eliminating the NPs on request; level 2: replace the NPs with a safer product; level 3: reduce exposure to NPs through the use of PPE or particular production procedures); (d) deciding whether to continue the development and production of nano material (sharing of information with stakeholders; collect more security information); and (e) update the risk assessment process through regular reviews. To better clarify, risk knowledge has been categorized into simple, complex, uncertain, or ambiguous, based upon whether the method of evaluation was scientific (evidence-based) or societal (value-based). Simple risks have clear cause–effect relationships for materials and their impact. Complex risk refers to the difficulty in identifying the causal links and their effects. There is insufficient knowledge about the cause and effect relationship. Uncertain risk knowledge refers to the incompleteness of knowledge, with the available knowledge relying on uncertain assumptions, assertions, and predictions. Ambiguous risk knowledge has variable interpretations, although it largely denotes a lack of proper understanding of the phenomena and their effects. Today the particle size distribution is one of the main physicochemical characteristics most studied in toxicological studies on NPs; however, other important parameters in which to concentrate research are: surface, surface reactivity, solubility in water, agglomeration, chemical composition, morphology, particle number, and mass concentrations [83]. According to the above, nanotechnology products will require pre-market testing for health and environmental impact, life cycle assessment, and consideration of secondary risks. Therefore, further studies will have to develop appropriate methodologies for monitoring nanomaterials together with methods to reduce exposure.

2.6  Control Banding for Engineered Nanoparticles The traditional approach to protecting workers’ health is based on measuring the exposure to potentially hazardous agents. Measurements of worker exposures to these agents are typically compared to occupational exposure limits (OELs) to determine if existing control measures provide adequate protection. Reliance on this approach has become increasingly difficult due to the growing number of potentially hazardous materials in the workplace that do not have OELs. The large and rapidly growing number of types and structures of nanomaterials (e.g.,

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nanoparticles, nanofibers, nanotubes) has presented a major challenge as it is impossible to perform toxicological evaluation on each nanomaterial prior to potential worker exposure. Control banding (CB) is a risk management strategy that has been used to identify and recommend exposure control measures to potentially hazardous substances for which toxicological information is limited. A number of different strategies have been proposed for using CB in workplaces where exposure to engineered nanomaterials can occur. At the base of the CB there is the awareness that there is really only a limited number of approaches for risk control and management, that had been developed by solutions previously used to control similar professional exposures: (1) potential hazard control, (2) engineering controls, (3) good professional hygiene practices, and (4) advices from qualified personnel. A basic principle for CB is the requirement for an easy-to-use method, even for non-expert, capable of giving consistent and accurate results, as a possible alternative to complicated sampling. Appropriate controls, based on hazard and exposure bands, must be correctly implemented and managed through periodic evaluations, verifying their correct and safe functioning through monitoring, in order to keep workers’ exposures within acceptable limits. Control banding does not replace experts in occupational safety and health area, nor it does eliminate the need for exposure monitoring. In conclusion, the use of CB for reducing exposures to nanomaterials has the potential to be an effective risk management strategy when information is limited on the health risk to the nanomaterial and/or there is an absence of an OEL. However, there remains a lack of evidence to conclude that the use of CB can provide adequate exposure control in all work environments. Additional validation work is needed to provide more data to support the use of CB for the safe handling of nanomaterials.

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Chapter 3

Immune Toxicity of and Allergic Responses to Nanomaterials Yasuo Yoshioka, Toshiro Hirai, and Yasuo Tsutsumi

Abstract  Over the past decade, the remarkable development of nanomaterials and nanotechnology has led to their use in many applications. But as the uses of nanomaterials have increased, so have concerns regarding their potential adverse effects (that is, nanotoxicity) in humans and the environment. Because the body’s immune systems are responsible for dealing with foreign substances, we likely should expect at least some interaction of nanomaterials with our immune systems with daily use of nanomaterials, and we must understand those interactions in order to use nanomaterials safely or to develop safer nanomaterials. In this review, we summarize recent advances in immunotoxicology studies of nanomaterials, especially (1) macrophage recognition of nanomaterials with particular emphasis on the effect of par-

Y. Yoshioka (*) BIKEN Innovative Vaccine Research Alliance Laboratories, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan BIKEN Center for Innovative Vaccine Research and Development, The Research Foundation for Microbial Diseases of Osaka University, Osaka, Japan Laboratory of Nano-Design for Innovative Drug Development, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan The Center for Advanced Medical Engineering and Informatics, Osaka University, Osaka, Japan e-mail: [email protected] T. Hirai Departments of Dermatology and Immunology, University of Pittsburgh, Pittsburgh, USA Y. Tsutsumi The Center for Advanced Medical Engineering and Informatics, Osaka University, Osaka, Japan Laboratory of Toxicology and Safety Science, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan © Springer Nature Singapore Pte Ltd. 2020 T. Otsuki et al. (eds.), Allergy and Immunotoxicology in Occupational Health - The Next Step, Current Topics in Environmental Health and Preventive Medicine, https://doi.org/10.1007/978-981-15-4735-5_3

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ticle size, and (2) in vivo responses after skin exposure to nanomaterials, including the onset or aggravation of allergy. In addition, we discuss challenges to further understanding the immune system–nanomaterial interaction, with the goal of increasing the safety of these compounds. Keywords  Allergy · Cytokine · Nanomaterial · Nanotoxicity · Macrophage · Skin

3.1  Introduction Recent advances in nanotechnology have enabled the design and production of many engineered nanomaterials (i.e., materials with at least one dimension measuring 1–100 nm), including nanoparticles, nanofibers, and nanosheets. Due to their small size and large surface area, nanomaterials have unique physical and chemical properties, such as enhanced hardness, plasticity, conductivity, diffusivity, optical properties, and chemical reactivity, compared with larger materials, making nanomaterials attractive in a wide range of applications. More than 1800 diverse nanotechnology-­based products have been used in a broad range of applications in the machinery and textile industries, cosmetics, and medicine. As the use of nanomaterials increases, so have concerns regarding the safety of nanomaterials; these concerns regarding nanotoxicity have arisen due to two main reasons [1]. The first reason is that, compared with larger materials, nanomaterials have greater potential to travel through an organism because they can cross various biologic barriers [2], for example, the placenta [3] and blood−milk barrier [4]. The second key reason is that the biologic activity of a material typically increases as its particle size decreases: smaller materials occupy less volume, leading to increased surface area per unit mass and thus increased potential for biologic interaction. In accordance with these factors, both in vitro and in vivo studies as well as both acute and chronic experimental models have revealed previously unappreciated toxic effects of nanomaterials, including immunotoxicity, neurotoxicity, genotoxicity, and reproductive toxicity. In addition, concerns regarding the human health risks of engineered nanomaterials have emerged [5, 6]. Therefore, more information regarding nanotoxicity is warranted for utilizing the potential benefits of nanomaterials and to design safer nanomaterials. Given that—like bacteria, viruses, and allergens—nanomaterials are foreign substances to the body, they likely would first encounter the immune system and consequently trigger various immune responses. Therefore, because nanomaterials probably initiate some sort of an immune response regardless of the route of exposure, the immunotoxicity of nanomaterials is one of the highest priority research interests in the nanotoxicity field [7, 8]. Two predominant scenarios of immunotoxicity are: (1) nanomaterials induce an inflammatory response that directly causes a health problem and (2) the immune response induced through exposure to nanomaterials modulates immune responses to other targets, consequently deranging

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appropriate responses to targets (e.g., suppressing immune response to viruses) or aggravating ongoing health issues (e.g., exacerbating allergy). To prevent either situation, we need to understand the mechanisms through which our immune systems recognize nanomaterials and the various possible responses to this recognition. In this review, we overview current knowledge regarding immune recognition of nanomaterials and their hazards in vivo.

3.2  Nanomaterial-Recognizing Receptors on Macrophages Professional phagocytes such as macrophages, dendritic cells, and neutrophils are primarily responsible for the elimination of foreign substances from the body. Therefore, these cells likely are similarly charged with recognizing nanomaterials. Once phagocytes find nanomaterials, the immune response induced probably depends on how they are recognized. For example, some nanomaterials might induce macrophage activation because they are recognized by immune-activating receptors and consequently trigger the production of inflammatory cytokines and chemokines, thereby recruiting numerous inflammatory immune cells and thus resulting in tissue damage. We begin this discussion by introducing reports regarding the receptors on macrophages that potentially recognize nanomaterials. A wide variety of cell-surface molecules on macrophages have been shown to be receptors for pathogenic particles. For example, class A scavenger receptors, such as SR-A1 and MARCO, and class B scavenger receptors, such as SR-B1 and CD36, bind to bacteria and apoptotic cells [9], indicating that these molecules might be candidate cellular receptors of nanomaterials. In fact, polystyrene nanoparticles and SiO2 nanoparticles bind to MARCO [10, 11]. SR-A1 and the mannose receptor CD206 contribute to the uptake of SiO2 particles by primary human macrophages [12]. In addition, SR-B1 is a receptor for SiO2 nanoparticles, and SR-B1-deficient macrophages neither internalized SiO2 nanoparticles nor promoted inflammatory responses to SiO2 nanoparticles. Furthermore, SR-B1 binds to SiO2 nanoparticles but not TiO2 nanoparticles [13], even though SR-A1 and MARCO are known to bind to both SiO2 and TiO2 particles [14]. Regarding their immunotoxicities, one characteristic point of nanomaterials needs to be remembered. Nanomaterials can be quite different in size, shape, and surface charge even when their core materials have the same chemical formula (Fig. 3.1). That is, even nanomaterials with the same name (e.g., SiO2 nanoparticles) can behave differently depending on the specific product in which they are used, and the immune system may recognize and therefore respond to these formulations differently. Many recent studies including our own have shown that size, shape, and surface charge all influence the pro-inflammatory effects of nanomaterials [1, 15]. For example, when we compared the inflammatory effects of SiO2 particles that differed in diameter (30–1000 nm), SiO2 nanoparticles with diameters of 30 and 70 nm induced greater cytokine production by macrophages in vitro than did larger particles [16]. Furthermore, intraperitoneal injection of SiO2 nanoparticles induced

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Macrophages

NLRP3

IL-1β

Receptor-dependent size effect

Size dependent intracellular behavior

Receptor-independent size effect

Fig. 3.1  Several parameters contribute to the immunotoxicity of nanomaterials

stronger inflammatory responses with cytokine production than did larger particles, whereas surface modification of SiO2 nanoparticles suppressed inflammatory responses. However, the mechanism underlying these differences (such as whether the recognizing receptor changes) largely remains to be clarified.

3.3  S  ize-Specific Effects of Nanomaterials on Their Recognition by Macrophages We recently investigated the effect of the size of SiO2 particles (diameter: 10, 30, 50, 70, 100, 300, and 1000  nm) on pro-inflammatory responses by a human macrophage cell line [17]. The secretion of IL-1β showed a bell-shaped distribution, where SiO2 nanoparticles with a diameter of 50 nm had the greatest effect on secretion; SiO2 particles with larger or smaller diameters had progressively less effect on IL-1β secretion by the cell line. Interestingly, SR-A1 contributed to IL-1β induction

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and the cellular uptake of 50- and 100-nm SiO2 nanoparticles but not their 10- and 1000-nm counterparts; this results suggest that only SiO2 nanoparticles of specific sizes induce SR-A1-mediated inflammatory responses. In addition, after intravenous injection of mice with SiO2 nanoparticles that ranged in size from 10 to 1000 nm [18], decreases in platelet count and increases in liver damage and lethal toxicity showed a simple correlation with decreasing particle size; in contrast, among the variously sized SiO2 nanoparticles, those 50 nm in diameter induced the most severe hypothermia. These results show that the inflammatory responses induced by nanomaterials can be highly size-specific and that various nanomaterial-­ recognizing receptors critically regulate these responses. Several in  vitro studies have shown that cellular uptake of nanomaterials is most efficient for those with a diameter with 50 nm, compared with larger and smaller nanomaterials [19, 20], thus supporting the concept of size-specific immunotoxicity. Taken together, differences in the recognition mechanism, such as the receptors involved, contribute to the size-­ associated effect on the pro-inflammatory response to nanomaterials (Fig. 3.1). Macrophage subsets differ in the efficiency with which they uptake nanomaterials. For example, 300 nm particles are cleared more slowly in Th1-prone mice compared with Th2-prone mice, and M2 macrophages, which are induced by Th2 cytokines, take up nanoparticles more efficiently than M1 macrophages [21]. In addition, M2 polarization of macrophages promotes nanoparticle internalization [22], suggesting that global immune regulation and macrophage subsets should be considered in regard to the clearance and immunotoxicity of nanomaterials. However, whether the different responses by various macrophage subsets are due to the presence of different nanomaterial-recognizing receptors remains to be seen.

3.4  P  otential Phagocytic Receptor Independent Size Effect of Nanoparticles Among the inflammatory responses induced by nanomaterials, the NLRP3 inflammasome-­mediated pro-inflammatory effects of nanomaterials, such as the production of IL-1β (a strong pro-inflammatory cytokine), are gaining particular attention because of their role in initiating inflammation [23]. The secretion of IL-1β is tightly regulated and involves at least two processes: the NF-κB-dependent synthesis of pro-IL-1β and the NLRP3 inflammasome (caspase 1)-dependent cleavage of pro-IL-1β for the secretion of mature IL-1β. NLRP3-deficient macrophages do not secrete IL-1β after their treatment with nanomaterials, indicating that activation of the NLRP3 inflammasome is indispensable for nanomaterial-induced IL-1β secretion [23]. Both SiO2 and TiO2 nanoparticles strongly activate macrophages to induce IL-1β secretion in vitro and promote pulmonary inflammation in vivo [24]. In addition, NADPH oxidase-dependent activation of the NLRP3 inflammasome is crucial for the lung fibrosis due to multiwalled carbon nanotubes [25]. It is thought that IL-1β from macrophages acts on pulmonary epithelial cells and fibroblasts,

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stimulating these cells to produce various inflammatory cytokines, including TNF-α and IL-6, and resulting in massive inflammation in the lung [26]. Several studies have demonstrated that K+ efflux, lysosomal stress, and reactive oxygen species are involved in the activation of the NLRP3 inflammasome by micro- and nanomaterials [23]. In addition, carbon black nanoparticles were shown to induce the programmed cell death designated pyroptosis, which is distinct from apoptosis, through the NLRP3 inflammasome [27]. We noted that 50 nm and 100 nm SiO2 nanoparticles that are both acquired through scavenger receptor A1, while 50 nm achieve the greater activation of NLRP3 to induce production of IL-1β compared to 100 nm in an independent way of the amount of silica particles taken up by the cells, suggesting that only specific size of particles activate intracellular signaling that is somehow coupled with the receptors on phagocytic cells [17]. We also noted that differences in particle size could influence membrane trafficking of endosomal vesicles, thus suggesting that the size effect of nanomaterials can be induced in a receptor-independent manner [28] (Fig. 3.1). The fact that some crystals can induce lysosomal damage independently of phagocytic receptors, which activate NLRP3 to induce IL-1β, might support this hypothesis [29].

3.5  I mmune Responses Due to Cutaneous Exposure to Nanomaterials Because of the potential for inhalational exposure to nanomaterials in the workplace, in  vivo studies have particularly focused on the pulmonary inflammatory responses to these compounds [30]. However, the increasing use of nanomaterial-­ containing skin care and other consumer products provides many opportunities for dermal exposure to nanomaterials [31]. For example, because their ultraviolet-­ protective properties are stronger than those of larger particles, ZnO and TiO2 nanoparticles have been used in sunscreens. SiO2 nanoparticles are used as an anti-­ setting agent in a wide variety of cosmetics, and Ag nanoparticles (because of their antimicrobial properties) are found in diverse consumer products, including clothing, antibacterial sprays, detergent, socks, and shoes. The stratum corneum prevents the penetration of some foreign substances into the body, and, in general, healthy skin is considered to be impervious to all but small (